Method of purifying calcium ion-binding protein

ABSTRACT

The present invention relates to a method for purifying a calcium ion-binding protein by cation exchange chromatography. The present invention provide a method for isolating and purifying a calcium ion-binding protein in a simple and efficient manner from a liquid sample containing a calcium ion-binding protein and contaminants without any pretreatment such as addition of a chelating agent. More specifically, the present invention relates to a method for purifying a calcium ion-binding protein which comprises contacting said protein with a cation exchange carrier in the presence of calcium ions to let the said protein be adsorbed to the carrier, and after washing, eluting said protein, and to a calcium ion-binding protein having substantially no contaminants obtained by the method of the present invention.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a method for purifying a calciumion-binding protein by a cation exchange process. More specifically, thepresent invention relates to a method for purifying a calciumion-binding protein which comprises contacting said protein with acation exchange carrier in the presence of calcium ions to render saidprotein be adsorbed to the cation exchange carrier, and after washing,eluting said protein from the cation exchange carrier, and to a calciumion-binding protein obtained by said method which contains substantiallyno contaminants.

BACKGROUND OF THE INVENTION

[0002] For isolation and purification of a protein of interest fromcontaminants, physico-chemical properties such as a molecular size, anelectric charge on the surface or solubility of said protein isutilized. A process for purification commonly used in the field ofprotein chemistry includes, for instance, salting out, ultrafiltration,isoelectric precipitation, electrophoresis, ion exchange chromatography,gel filtration chromatography, affinity chromatography, and the like. Incase that a protein of interest must be purified from the livingtissues, cells, or blood, where an enormous variety of differentproteins exist, these processes need often be combined in a manifoldmanner. However, it is possible to provide a method for purificationwith much more specificity by utilizing a property commonly shared by acertain kind of protein.

[0003] By way of example, a unique method for purification using anionexchange chromatography is known wherein a divalent cation-bindingprotein is adsorbed to an anion exchange resin and then eluted therefromwith a divalent cation to specifically purify said divalentcation-binding protein as disclosed in Japanese patent publication No.200180/1990. According to this method, a chelating reagent such asethylenediaminetetraacetic acid (EDTA) is added to a solution containinga divalent cation-binding protein to first remove divalent ions. Then,the resulting solution is contacted with an anion exchange resin such asMonoQ to render the divalent cation-binding protein be adsorbed to theanion exchange resin. Finally, addition of sodium chloride and calciumchloride elutes the divalent cation-binding protein from the anionexchange resin. However, most of naturally occurring proteins arenegatively charged under physiological conditions and hence numerouscontaminants other than a protein of interest are preferentiallyadsorbed to an anion exchange resin, thus hampering efficientpurification of the desired protein. Therefore, this method forpurification through adsorption of a desired protein to an anionexchange resin is preferably used for a small amount of a proteinsolution or at an advanced stage of purification processes.

[0004] Japanese patent publication No. 258286/1995 discloses a methodfor purifying a calcium ion-binding, vitamin K-dependent protein by ananion exchange process wherein calcium chloride is added to a solutioncontaining a vitamin K-dependent protein and the resulting solution ispassed through an anion exchange resin to isolate the desired proteinfrom contaminating proteins. This method, however, is disadvantageous inthat a large volume of fractions containing the desired protein must bepassed through and hence subsequent procedures will become troublesomeespecially when conducted in a large scale.

[0005] Annexin V, one of calcium ion-binding proteins, is a simpleprotein of about 34 kDa molecular weight bearing no sugar chain that hasa physiological activity such as anti-coagulating activity, cornealepithelium-extending activity, and phospholipase A₂ inhibitory activity.It is known that Annexin V distributes in a variety of tissues andsecretions within the living body including human placenta (Chem.Pharma. Bull., 38, 1957-1960, 1990). Annexin V is called a calciumion-binding protein since it has an ability to bind with a lipidmembrane via calcium ions.

[0006] Annexin V has been extracted from organs of human or animals(Japanese patent publication No. 174023/1987). Nowadays, however, it canbe produced in E. coli and yeast by the use of the genetic recombinationtechnique (Japanese patent publications No. 20095/1989 and No.219875/1991).

[0007] Annexin V has conventionally been purified, after pretreatment ofan Annexin V-containing solution with precipitation, membrane filtrationand centrifugation, by a combination of ammonium sulfate fractionation,anion exchange chromatography, hydrophobic chromatography and affinitychromatography (Jurgen Romisch et al., Biochem. J. 272, 223-229, 1990;T. R. Hawthorne et al., Journal of Biotechnology 36, 129-143, 1994).

DISCLOSURE OF THE INVENTION

[0008] However, these processes are disadvantageous in that purificationsteps are complicated and troublesome requiring a great deal of laborand time and hence possibly meet an obstacle in view of reproducibilityand yield, rendering them not be suitable for purification of Annexin Vin an industrial scale. Moreover, as purification process in a largescale, these processes are disadvantageous in economical point of viewas well since they used heparin Sepharose, which is rather expensive,for enhancing purification degree of Annexin V. Previously, the presentinventors have provided a method for preparing Annexin V by pretreatinga protein solution to remove contaminants to some extent and thenperforming anion exchange chromatography on the resulting solution(Japanese patent publication No. 219875/1991). This method mightpossibly enables purification of Annexin V in an industrial scale butwould not exceed the method of the present invention.

[0009] As described above, for use in an industrial scale, theconventional processes are problematic in view of cost, efficiency andhandling.

[0010] An object of the present invention is to provide a method forisolating and purifying a calcium ion-binding protein in a simple andefficient manner from a liquid sample containing a calcium ion-bindingprotein and contaminants without any pretreatment such as addition of achelating agent.

[0011] Another object of the present invention is to provide Annexin Vof high purity obtained by the method of the present invention.

[0012] Under the circumstances, the present inventors investigated forattaining the above objects and have found that Annexin V, one ofcalcium ion-binding proteins, is adsorbed to SP-Sepharose cationexchange carrier in the presence of calcium chloride and at pH of aroundneutrality. The present inventors have also noted that the adsorption ofAnnexin V to SP-Sepharose cation exchange carrier occurred specificallyin the presence of calcium ions but could scarcely observe theadsorption in the presence of other divalent ions than calcium ions,e.g. magnesium ions. With this finding, the present inventors addedcalcium chloride to a large amount of homogenate of Annexin V-producingcells produced by the genetic recombination technique and contacted theresulting homogenate with SP-Sepharose cation exchange carrier which hasbeen equilibrated with ammonium chloride buffer containing calciumchloride. After washing, elution was performed by decreasing or removingcalcium chloride level or with ammonium chloride buffer containingsodium chloride in the presence of calcium ions to successfully purifyAnnexin V with high purity. Moreover, a trace amount of remainingproteases could successfully be removed by performing said cationexchange chromatography at pH 9.0.

[0013] Thus, the present invention encompasses a method for purificationof a calcium ion-binding protein, either naturally occurring or producedby the genetic recombination technique, by cation exchangechromatography using SP-Sepharose cation exchange carrier in thepresence of calcium chloride.

[0014] The present invention also encompasses a calcium ion-bindingprotein, either naturally occurring or produced by the geneticrecombination technique, thus obtained by the method of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIGS. 1A and 1B are a schematic illustration of cloning of AnnexinV structural gene and preparation of yeast cells transformed with saidgene.

[0016]FIG. 2 shows results of gel filtration chromatography for (a)samples prior to cation exchange chromatography, (b) fractions passedthrough cation exchange chromatography, and (c) fractions eluted fromcation exchange chromatography after washing.

[0017]FIG. 3 shows results of gel filtration chromatography forfractions eluted from cation exchange chromatography after washing.

[0018]FIG. 4 shows an elution pattern of Annexin VI from SP-Sepharose.

[0019]FIG. 5 is a photograph showing results of SDS-PAGE of Annexin VI.Lane 1: BioRad prestained marker proteins; phosphorylase B (116,000),BSA (80,000), ovalbumin (52,500), carbonic anhydrase (34,900), soybeantrypsin inhibitor (29,900), lysozyme (21,800); Lane 2: Annexin VIstandard; Lane 3: elution with DEAE-Toyopearl (sample); Lane 4:SP-Sepharose, fraction No. 5; Lane 5: fraction No. 7; Lane 6: fractionNo. 9; Lane 7: fraction No. 16; Lane 8: fraction No. 51; Lane 9:fraction No. 54; and Lane 10: fraction No. 57.

[0020]FIG. 6 shows an elution pattern of coagulation factor X fromSP-Sepharose.

[0021]FIG. 7 is a photograph showing results of SDS-PAGE of coagulationfactor X. Lane 1: BioRad prestained marker proteins; phosphorylase B(116,000), BSA (80,000), ovalbumin (52,500), carbonic anhydrase(34,900), soybean trypsin inhibitor (29,900), lysozyme (21,800); Lane 2:commercially available coagulation factor X; Lane 3: fraction No. 4;Lane 4: fraction No. 7; Lane 5: fraction No. 11; and Lane 6: fractionNo. 12.

BEST MODE FOR CARRYING OUT THE INVENTION

[0022] The method of the present invention comprises a step in which aliquid sample containing a calcium ion-binding protein is contacted witha cation exchange carrier in the presence of calcium ion, followed by astep in which a concentration of calcium ion is decreased or removedand/or a concentration of counter ions (salts) is increased to elute andrecover said protein. The method of the present invention enablesproduction of a calcium ion-binding protein with high purity. Thecontacting process with the cation exchange carrier may be performedeither in a batch or by chromatography. When chromatography is used, acolumn size may appropriately be selected depending on a productionscale.

[0023] A cation exchange carrier used herein includes, but not limitedto, SP-Sepharose, CM-Sepharose, CM-cellulose, SE-cellulose, S-Spherodex,SP-Spherosil, and the like, all of which are commercially available.Among these, SP-Sepharose is preferably used.

[0024] An amount of a protein solution to be contacted with the carriermay vary depending on a concentration of the solution or an ability ofthe carrier for adsorption. In case of SP-Sepharose, for instance, 0.1to 30 g/L carrier of the protein may be used. Preferably, 15 to 20 g/Lcarrier of the protein is used.

[0025] A flow rate while adsorption to the cation exchange carrier maybe 1 to 150 cm/h, preferably 15 to 100 cm/h, more preferably 50 to 80cm/h. On the other hand, a flow rate while elution of the adsorbedprotein from the cation exchange carrier may be 1 to 150 cm/h,preferably 30 to 100 cm/h, more preferably 30 to 80 cm/h.

[0026] A buffer that may be used for adsorption to or elution from thecation exchange carrier includes any buffer conventionally used in ionexchange chromatography, including ammonium chloride buffer, citratebuffer, acetate buffer and Tris-HCl buffer. Among these, ammoniumchloride buffer is preferably used. A concentration of a buffer may bein a range of 5 to 100 mM, preferably 10 to 40 mM. A buffer may be usedat pH 5 to 10, preferably at pH 8 to 9.5, conditions where proteases areremoved. More preferably, 20 mM (around pH 9.0) ammonium chloride bufferis used.

[0027] As a source of calcium ions, any substance that can affordcalcium ions may be used, including calcium chloride, calcium carbonate,preferably calcium chloride.

[0028] When a large quantity of calcium chloride is added to homogenateof tissues or cells or plasma, hydrophobic proteins or high molecularweight compounds are sometimes deposited as a result of salting-out.Thus, calcium ions may preferably be used in such an amount that notonly renders a calcium ion-binding protein be bound to and isolated fromthe cation exchange carrier but also forms no precipitation from aliquid sample containing a calcium ion-binding protein.

[0029] For adsorption of a calcium ion-binding protein to the cationexchange carrier, calcium ions at a concentration of 5 to 100 mM maypreferably be used. More preferably, calcium ions at a concentration of10 to 30 mM may be used. In combination with a buffer to be used foradsorption to and elution from the cation exchange carrier, 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride maypreferably be used.

[0030] The adsorbed calcium ion-binding protein may be eluted from thecarrier by removing or decreasing calcium ion level in the buffer oradding other counter ions than calcium ions, or both. A counter ionincludes Na⁺, Li⁺, K⁺ ions, and the like. Preferably, elution may beperformed by adding 1 to 500 mM, more preferably 50 to 500 mM, stillmore preferably 50 to 300 mM sodium chloride to the ammonium chloridebuffer, and most preferably by adding 200 mM sodium chloride to 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride.Alternatively, the adsorbed calcium ion-binding protein may be elutedfrom the carrier merely by decreasing the calcium chloride level to lessthan 5 mM.

[0031] The method of the present invention, even when used solely, canafford to provide purification of a calcium ion-binding protein of 80%purity or more. It may more efficiently be used, however, in combinationwith other purification processes. For example, in case that a liquidsample containing a calcium ion-binding protein is contaminated withinsoluble substances, pretreatment for removing such substances, e.g.centrifugation, salting-out, membrane filtration, etc., is preferablycarried out prior to the method of the present invention.

[0032] In addition to the above-described processes, other purificationprocesses of various chromatographic procedures, including anionexchange chromatography, hydrophobic chromatography, gel filtrationchromatography, affinity chromatography, adsorption chromatography, etc.may be performed together with the method of the present invention toprovide a calcium ion-binding protein of higher purity. The method ofthe present invention may be used at any stage of the above-describedprocesses. Preferably, after a sample containing a calcium ion-bindingprotein is pretreated to remove insoluble substances, the method of thepresent invention is used and then anion exchange chromatography isfollowed. More specifically, an Annexin V-containing fraction obtainedby the cation exchange chromatography is applied to Q-Sepharose columnequilibrated with 10 mM sodium phosphate buffer (pH 7.4) containing 50mM sodium chloride, and after washing, elution is performed with lineargradient of concentration from 50 mM to 500 mM sodium chloride to giveAnnexin V with much higher purification.

[0033] A calcium ion-binding protein to be purified by the method of thepresent invention typically includes Annexins I, II, III, IV, V, VI andVII but may be any protein that has an ability to bind to calcium ions,such as coagulation factor X.

[0034] The method of the present invention may efficiently be applied toblood, body fluid and tissue homogenate from an animal either naturallyoccurring or genetically engineered that produces a calcium ion-bindingprotein, as well as cell homogenate and culture supernatant ofrecombinant cells, including plant cells, bacterial cells, yeast cells,insect cells and animal cells. preferably, the method of the presentinvention may be used in recombinant yeast cells producing a calciumion-binding protein. More preferably, the method of the presentinvention may be used in cell homogenate or culture supernatant of yeastcells producing Annexin V.

[0035] Annexin V thus prepared, having special physiological activities,may be formulated into a pharmaceutical preparation in any conventionaldosage form such as injections, eye drops, oral preparations,suppositories, etc. alone or in combination with a pharmaceuticallyacceptable carrier, diluent, stabling agent or preservative.

[0036] According to the present invention, an efficient method forpurifying a calcium ion-binding protein with high purity is provided.Also provided is the calcium ion-binding protein thus obtained by themethod of the present invention having substantially no contaminants.

[0037] According to the method of the present invention, most proteinsnegatively charged under physiological conditions are passed through thecation exchange carrier whereas a calcium ion-binding protein, which canform a complex with calcium ions, is preferentially adsorbed to thecarrier. Thus, the method of the present invention enables handling of alarge quantity of a sample at one time without deterioration of theadsorption capacity of the cation exchange carrier by contaminatingproteins other than the desired protein.

EXAMPLE Preparation Example Preparation of Recombinant Yeast CellProducing Annexin V

[0038] Recombinant yeast cells producing Annexin V were prepared asdescribed in a publication of patent application (Japanese PatentPublication No. 219875/1991). FIG. 1 schematically shows preparation ofthe recombinant yeast cells wherein the term “CPB-I” is used forreferring to “Annexin V”.

[0039] (1) Cloning of Annexin V Structural Gene

[0040] From human placenta cDNA library (Clontech Laboratories, Inc.),phage that bears Annexin V structural gene was insolated byimmunoscreening using anti-Annexin V monoclonal antibody. DNA was thenprepared from phage and digested with restriction enzyme EcoRI toproduce a fragment, which was then inserted into the EcoRI site ofpUC118 vector to construct pMKT7.

[0041] (2) Construction of Expression Plasmid

[0042] The plasmid pMKT7 was digested with restriction enzymes NcoI andSacI and a DNA fragment containing Annexin V structural gene wasseparated by agarose electrophoresis. Addition of a synthetic linkerconverted both ends of the DNA fragment into XhoI and BamHI sites. Theresulting DNA fragment was inserted into XhoI and BamHI sites of theexpression vector pPS1 to construct expression vector pAPCPBI.

[0043] (3) Preparation of Recombinant Yeast Cells

[0044] Host yeast cells (Saccharomyces cerevisiae AH22) were transformedwith the expression plasmid pAPCPBI by the lithium acetate technique.After transformation, colonies appeared on an agar medium deprived ofleucine were isolated and an expression level was measured. Those cloneswith higher expression level were selected and subjected to repetitionof plating to the agar medium, isolation of colonies and measurement ofexpression level to give recombinant yeast cells with stability.

Example 1 Purification of Recombinant Annexin V

[0045] (1) Culture of Annexin V-Producing Recombinant Yeast Cells

[0046] Annexin V-producing recombinant yeast cells were cultured on 2Lsynthetic selection medium at 28° C. for 3 days. The recombinant yeastcells were then inoculated to 88L selection medium and cultured at 28°C. for 2 days. The recombinant yeast cells were then transferred to 810Lsemisynthetic medium (40 g sucrose, 5 g yeast extract, 5 g ammoniumsulfate and 0.5 g magnesium sulfate septahydrate in 1L medium) andculture was continued at 28° C. for 24 hours.

[0047] (2) Pretreatment of Annexin V in Large Quantity Prior toPurification

[0048] The large culture solution was filtered with a 0.1 μm membranefilter to collect the recombinant yeast cells, which were physicallyruptured with a French press-type cell homogenater. The ruptured cellsuspension was filtered with the membrane filter and the filtrate wasconcentrated with a ultrafiltrater. To the concentrate was added aceticacid for isoelectric precipitation (pH 5.0). Precipitates formed werefiltered with the membrane filter to remove the precipitates. Then, pHof the filtrate was adjusted to 9.0 with ammonia and the filtrate wasagain concentrated with a ultrafiltrater (pretreated solution).

[0049] (3) Cation Exchange Chromatography (Elution by Decreasing orRemoving Calcium Chloride Level)

[0050] To the pretreated solution was added a calcium chloride solutionto a final concentration of 20 mM and was subjected to cation exchangechromatography with SP-Sepharose (Pharmacia). Specifically, thepretreated solution supplemented with calcium chloride was applied to acolumn equilibrated with 20 mM ammonium chloride buffer (pH 9.0)containing 20 mM calcium chloride and 50 mM sodium chloride. Afterwashing with the buffer, the column was further washed with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride.Then, Annexin V was eluted with 20 mM ammonium chloride buffer (pH 9.0).

[0051] (4) Cation Exchange Chromatography (Elution by Increasing SodiumChloride Level)

[0052] As in the step (3), the pretreated solution was added with acalcium chloride solution to a final concentration of 20 mM and wassubjected to cation exchange chromatography with SP-Sepharose.Specifically, the pretreated solution supplemented with calcium chloridewas applied to a column equilibrated with 20 mM ammonium chloride buffer(pH 9.0) containing 20 mM calcium chloride and 50 mM sodium chloride.After washing with the buffer, Annexin V was eluted by a linear gradientof concentration of sodium chloride from 50 mM up to 300 mM with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride(flow rate at adsorption and elution: 56.7 cm/h).

[0053]FIG. 2 shows elution patterns obtained by gel filtrationchromatography of (a) sample prior to cation exchange chromatography,(b) fractions passed through cation exchange chromatography, and (c)fractions eluted from cation exchange chromatography, respectively. Gelfiltration chromatography was performed wherein 20 μL sample was appliedto TSKgel G3000 SW×1 (7.8 mm (ID)×30 cm) equilibrated with 10 mMphosphate buffer (pH 7.2) containing 0.14 M NaCl at a flow rate of 125.6cm/h. The results of gel filtration chromatography for fractions elutedfrom cation exchange chromatography are shown in Table 1 wherein puritywas obtained from the elution pattern.

[0054] (5) Anion Exchange Chromatography (Comparison with ConventionalTechnique)

[0055] Anion exchange chromatography was performed for the pretreatedsolution. The pretreated solution was applied to Q-Sepharose (Pharmacia)column equilibrated with 10 mM sodium phosphate buffer (pH 7.4)containing 50 mM sodium chloride. After washing, Annexin V was eluted bya linear gradient of concentration of sodium chloride from 50 mM up to300 mM. The results of gel filtration chromatography for the elutedfractions are shown in Table 1.

[0056] Annexin V obtained by the conventional technique and thatobtained by the method of the present invention were measured for theiranti-coagulating activity after further purification with additionalpurification processes. Measurement of anti-coagulating activity wasmade in the same manner as the quantification of sodium heparindescribed in the Japanese Pharmacopoeia (the 13th revision, p.900-901).Anti-coagulating activity was calculated wherein prolongation in timefor coagulation induced by 1 mg of standard sample (Annexin V purifiedby the conventional technique) was defined as one unit (U). As a result,no inactivation of Annexin V obtained by the method of the presentinvention was observed (Table 1). TABLE 1 Anion exchange Cation exchangechromatography chromatography Purity (%) 81.86 100 Activity (U/mg) 0.9to 1.0 0.9 to 1.0

Example 2 Purification of Recombinant Annexin V

[0057] A recombinant Annexin V was purified as in Example 1 except that20 mM citrate buffer (pH 6.0) containing 20 mM calcium chloride and 50mM sodium chloride was used in place of 20 mM ammonium chloride buffer(pH 9.0) containing 20 mM calcium chloride and 50 mM sodium chloride,and 20 mM citrate buffer (pH 6.0) was used in place of a linear gradientconcentration of sodium chloride from 50 mM up to 300 mM with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride, inthe cation exchange chromatography of the step (4). Flow rate was alsoaltered to 15.6 to 54.6 cm/h at adsorption and 39 cm/h at elution.

[0058]FIG. 3 shows an elution pattern obtained by gel filtrationchromatography of fractions eluted from cation exchange chromatography.

Example 3 Purification of Annexin VT from Placenta

[0059] One placenta (about 500 g) excepting the amnion and an umbilicalcord was sliced into pieces, washed with 2 L physiological saline, andminced with a meat grinder. After adding 400 mL of 50 mM Tris-HCl buffer(pH 7.4) containing 5 mM calcium chloride, 0.1% Triton X-100 and 5 mMbenzamidine, the mince was homogenated with a whirling blender. Thehomogenate was centrifuged at 10,000 rpm for 20 minutes to collectprecipitates, which were again suspended in 300 ml of 50 mM Tris-HClbuffer (pH 7.4) containing 50 mM EDTA and homogenated. The homogenatewas again centrifuged at 10,000 rpm for 20 minutes and an extract ofsupernatant was recovered (about 300 mL), to which 63 g ammonium sulfatewas added to prepare a 30%-saturated solution of ammonium sulfate. Aftercentrifugation to remove precipitates, to the supernatant was added 54 gammonium sulfate (60%-saturated ammonium sulfate) and precipitatedAnnexin VI fraction was recovered. Annexin V could preferentially berecovered in precipitates formed when the salt concentration was raisedto 80% saturation of ammonium sulfate.

[0060] The precipitates formed with a 60%-saturated solution of ammoniumsulfate were dissolved in 50 mM Tris-HCl buffer (pH 7.4) and dialyzedagainst the same buffer. A dialyzed solution (80 mL) was adsorbed toDEAE-Toyopearl (3×20 cm) equilibrated with the same buffer. Afterwashing with the same buffer, elution was performed by a linear gradientfrom the same buffer (180 mL) to 50 mM Tris-HCl buffer (pH 7.4)containing 0.3 M NaCl (180 mL)(each fraction: 4 mL/tube). Annexin VI ofinterest was eluted in fractions No. 46 to No. 50. These Annexin VIfractions were dialyzed against 20 mM ammonium chloride (pH 9.0), towhich was added calcium chloride to make finally 20 mM ammonium chloridebuffer (pH 9.0) containing 20 mM calcium chloride. The dialyzed solutionwas adsorbed to SP-Sepharose FF (1.5×8 cm) equilibrated with 20 mMammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride(FIG. 4; fractions No. 1 to No. 15). After washing with the same buffer,elution was performed with the same buffer supplemented with 0.5 M NaCl(FIG. 4; fractions No. 45 to No. 70). Samples at each stage wereanalyzed by non-reductive SDS-PAGE and the results are shown in FIG. 5.

Example 4 Purification of Coagulation Factor X

[0061] Commercially available coagulation factor X (about 1 mg) wasdialyzed against 20 mM Tris-HCl buffer (pH 8.0), to which was added onetenth amount of 200 mM calcium chloride to make finally 20 mM Tris-HClbuffer (pH 8.0) containing 20 mM calcium chloride. The dialyzed solutionwas adsorbed to SP-Sepharose FF (0.8×7 cm) equilibrated with 20 mMTris-HCl buffer (pH 8.0) containing 20 mM calcium chloride. Afterwashing with 20 mM Tris-HCl buffer (pH 9.0) containing 20 mM calciumchloride, elution was performed with 20 mM Tris-HCl buffer (pH 9.0)containing 0.3 M calcium chloride. In all stages, 5 mM benzamidine wasadded (Since benzamidine per se exhibits UV absorption at A280, anelution pattern is not explicit; FIG. 6). Fractions at each stage wereanalyzed by non-reductive SDS-PAGE and the results are shown in FIG. 7.

1. A method for purifying a calcium ion-binding protein from a samplecontaining said protein using a cation exchange carrier, wherein saidmethod comprises contacting the sample with the cation exchange carrierin the presence of calcium ions to let the protein be adsorbed to theexchange carrier; and eluting the adsorbed calcium ion-binding proteinfrom the exchange carrier by decreasing or removing a concentration ofcalcium ions and/or adding counter ions other than calcium ions.
 2. Themethod of claim 1 wherein the adsorption step is performed in thepresence of 5 to 100 mM calcium ions.
 3. The method of claim 2 whereinthe adsorption step is performed in the presence of 10 to 30 mM calciumions.
 4. The method of any one of claims 1 to 3 wherein the adsorptionstep is performed at a flow rate of 1 to 150 cm/h.
 5. The method ofclaim 4 wherein the adsorption step is performed at a flow rate of 15 to100 cm/h.
 6. The method of claim 4 wherein the adsorption step isperformed at a flow rate of 50 to 80 cm/h.
 7. The method of claim 1wherein the elution step is performed by decreasing a concentration ofcalcium ions to less than 5 mM.
 8. The method of claim 1 wherein theelution step is performed by adding 1 to 500 mM of counter ions otherthan calcium ions.
 9. The method of claim 1 wherein the elution step isperformed by adding 50 to 500 mM of counter ions other than calciumions.
 10. The method of claim 1 wherein the elution step is performed byadding 50 to 300 mM of counter ions other than calcium ions.
 11. Themethod of claim 1, 8, 9 or 10 wherein said counter ions are selectedfrom the group consisting of Na⁺, Li⁺ and K⁺.
 12. The method of claim 1,7, 8, 9, 10 or 11 wherein the elution step is performed at a flow rateof 1 to 150 cm/h.
 13. The method of claim 12 wherein the elution step isperformed at a flow rate of 30 to 100 cm/h.
 14. The method of claim 12wherein the elution step is performed at a flow rate of 30 to 80 cm/h.15. The method of claim 1 wherein the cation exchange carrier isselected from the group consisting of SP-Sepharose, CM-Sepharose,CM-cellulose, SE-cellulose, S-Spherodex and SP-Spherosil.
 16. The methodof any one of claims 1 to 15 wherein the calcium ion-binding protein isselected from the group consisting of Annexins I, II, III, IV, V, VI andVII.
 17. The method of any one of claims 1 to 16 wherein the samplecontains a calcium ion-binding protein prepared by the geneticrecombination technique.
 18. The method of any one of claims 1 to 17wherein the adsorption and elution steps are performed at pH 5 to 10.19. The method of claim 18 wherein the adsorption and elution steps areperformed at pH 8 to 9.5.
 20. The method of claim 19 wherein theadsorption and elution steps are performed at pH
 9. 21. The method ofclaim 1 wherein the adsorption step is performed in the presence of 10to 30 mM calcium ions at pH 8 to 9.5 at a flow rate of 15 to 100 cm/h;the elution step is performed at a flow rate of 30 to 80 cm/h bydecreasing a concentration of calcium ions to less than 5 mM or byadding 50 to 300 mM counter ions selected from the group consisting ofNa⁺, Li⁺ and K⁺; the cation exchange carrier is SP-Sepharose; thecalcium ion-binding protein is Annexin V; the sample contains Annexin Vprepared by the genetic recombination technique; and protease is removedfrom the sample.
 22. The method of claim 1 wherein the adsorption stepis performed in the presence of 10 to 30 mM calcium ions at pH 8 to 9.5at a flow rate of 15 to 100 cm/h; the elution step is performed at aflow rate of 30 to 80 cm/h by decreasing a concentration of calcium ionsto less than 5 mM or by adding 500 mM counter ions selected from thegroup consisting of Na⁺, Li⁺ and K⁺; the cation exchange carrier isSP-Sepharose; the calcium ion-binding protein is Annexin VI; the samplecontains naturally occurring Annexin VI; and protease is removed fromthe sample.
 23. A method for purifying a calcium ion-binding proteinfrom a sample containing said protein using a cation exchange carrier,wherein said method comprises contacting the sample with the cationexchange carrier in the presence of calcium ions to let the protein beadsorbed to the carrier.
 24. The method of claim 23 wherein said methodis performed in the presence of 5 to 100 mM calcium ions.
 25. The methodof claim 24 wherein said method is performed in the presence of 10 to 30mM calcium ions.
 26. The method of any one of claims 23 to 25 whereinsaid method is performed at a flow rate of 1 to 150 cm/h.
 27. The methodof claim 26 wherein said method is performed at a flow rate of 15 to 100cm/h.
 28. The method of claim 26 wherein said method is performed at aflow rate of 50 to 80 cm/h.
 29. The method of claim 23 wherein thecation exchange carrier is selected from the group consisting ofSP-Sepharose, CM-Sepharose, CM-cellulose, SE-cellulose, S-Spherodex andSP-Spherosil.
 30. The method of any one of claims 23 to 29 wherein thecalcium ion-binding protein is selected from the group consisting ofAnnexins I, II, III, IV, V, VI and VII.
 31. The method of any one ofclaims 23 to 30 wherein the sample contains a calcium ion-bindingprotein prepared by the genetic recombination technique.
 32. The methodof any one of claims 23 to 31 wherein the method is performed at pH 5 to10.
 33. The method of claim 32 wherein the method is performed at pH 8to 9.5.
 34. The method of claim 33 wherein the method is performed at pH9.
 35. A calcium ion-binding protein of high purity in a single peak asdetermined by gel filtration chromatographic analysis, obtained by themethod of any one of claims 1 to 34.